Streptococcus pneumoniae is a significant cause of morbidity and mortality in pediatric, geriatric, and immunocompromised populations. Until the mid-sixties, this pathogen was uniformly susceptible to penicillin, but since then the incidence of resistance to this antibiotic (as well as many others) has increased to such an extent that infections due to penicillin-resistant pneumococci have become a major threat in many countries. With regard to the USA, a nationwide survey showed that 5% of the pneumococci isolated in hospitals during the period 1979-'87 were intermediately resistant to penicillin (minimum inhibitory concentration (MIC) = 0.1-1 mg/L), and only 0.02% highly resistant (MIC>1 mg/L). Under the aegis of a Children's Hospital (Washington, D.C.) - NIDR clinical research training program, a pilot project was initiated to assess the prevalence of penicillin resistance among pneumococci isolated from patients attending this hospital. The one-year survey (from June 1992 through May 1993) showed that 8.3% of the 108 strains isolated were intermediately resistant to penicillin and 4.6% were highly resistant. Moreover, at least 40% of the penicillin-resistant strains were also resistant to frequently used oral and parenteral cephalosporins (cefaclor, cefixime, cefotaxime, cefpodoxime, cefuroxime, cephalexin, and loracarbef) and carbapenems (imipenem and meropenem). Also of great concern was the finding that all isolates were resistant to trimethoprim/sulfamethoxazole, a drug that is commonly used to treat infections in ambulatory patients. As a result of these findings, a study to define, at the sequence level, the molecular epidemiology and mechanism of trimethoprim resistance in S. pneumoniae was initiated. Initially, the trimethoprim-resistance determinant of several clinical isolates will be cloned, sequenced, and analyzed. Identification of specific residues responsible for resistance will also require the cloning and sequencing of the trimethoprim target protein from a sensitive strain. This cloning can be done by the PCR with primers based on sequences that are conserved in the resistant isolates. Alternatively, a library of the sensitive strain can be screened with gene probes derived from one or more resistant isolates. The information obtained from the comparative sequence analysis will be combined with modeling of the TMP-target protein to design novel antibiotics that will be effective against trimethoprim-resistant streptococci.